A quarter to a third of HIV-1 infected individuals eventually develop HIV-Associated Dementia (HAD). Neuronal injury and apoptosis may contribute to the cognitive deficits of HAD. In vitro and in vivo, the HIV-1 envelope glycoprotein gp120 produces injury and apoptosis in both human and rodent neurons, possibly via macrophage activation, and this may account for at least a part of the neuronal damage observed in HAD. Microglia, astrocytes and neurons express co-receptors for HIV-1, namely the chemokine receptors CCR5 and CXCR4. Using chemokine receptor knock-out mice, the objectives of this application are to further elucidate the role of microglia/macrophages in HIV-related neuronal damage, and to study the cellular signaling mechanisms affected by HIV-1 gp120 and chemokines in the brain. Such mechanisms may provide future therapeutic targets and thus new treatments for HAD. The following Specific Aims are proposed 1. To assess which chemokines and chemokine receptors are able to mediate or inhibit gp120-induced neuronal injury in mixed neuronal/glial mouse cerebrocortical cultures. 2. To assess if chemokine receptor activation on glial cells (macrophages/microglia or astrocytes), on neurons, or on both are necessary for neuronal apoptosis due to gp120. For the first two specific aims, both macrophage-tropic/R5 (CCR5- preferring) and T-cell tropic/X4 (CXCR4-preferring) gp120s will be tested in these experiments. To accomplish these aims, recombinant gp120 will be incubated in neuronal cultures produced from CXCR4(-/-) or CCR5(-/-) "knock out" mice in the presence and absence of wild-type microglia and astrocytes containing CXCR4 and CCR5 receptors. Alternatively, wild-type neurons will be incubated with microglia (or astrocytes) isolated from mice lacking these chemokine receptors. 3. To investigate in post mortem human brain tissue (from both demented and non-demented HIV-positive patients) if the appearance and concentration of distinct chemokines, such as SDF- 1 and fractalkine, are correlated with tissue damage and cognitive decline. Chemokines levels will be quantified by ELISA of brain extrancts. Additionally, potential signaling pathways will be assessed in microglia, astrocytes and neurons from the chemokine receptor knock-out mice (versus cells from wild-type mice); activation of the JNK, ERK and p38 mitogen-activated protein kinase pathways will be monitored by immunoblotting and immunocomplex protein kinase assays. Neuronal apoptosis due to gp120 or chemokines will be monitored using several approaches.